Antimicrobial resistance and heterogeneity of Neisseria gonorrhoeae isolated from patients attending sexually transmitted infection clinics in Lusaka, Zambia

Background Antimicrobial resistance (AMR) of Neisseria gonorrhoeae is a threat to public health as strains have developed resistance to antimicrobials available for the treatment of gonorrhea. Whole genome sequencing (WGS) can detect and predict antimicrobial resistance to enhance the control and prevention of gonorrhea. Data on the molecular epidemiology of N. gonorrhoeae is sparse in Zambia. This study aimed to determine the genetic diversity of N. gonorrhoeae isolated from patients attending sexually transmitted infection (STI) clinics in Lusaka, Zambia. Methods A cross-sectional study that sequenced 38 N. gonorrhoeae isolated from 122 patients with gonorrhea from 2019 to 2020 was conducted. The AMR profiles were determined by the E-test, and the DNA was extracted using the NucliSens easyMaG magnetic device. Whole genome sequencing was performed on the Illumina NextSeq550 platform. The Bacterial analysis pipeline (BAP) that is readily available at: https://cge.cbs.dtu.dk/services/CGEpipeline-1.1 was used for the identification of the species, assembling the genome, multi-locus sequence typing (MLST), detection of plasmids and AMR genes. Phylogeny by single nucleotide polymorphisms (SNPs) was determined with the CCphylo dataset. Results The most frequent STs with 18.4% of isolates each were ST7363, ST1921 and ST1582, followed by ST1583 (13%), novel ST17026 (7.9%), ST1588 (7.9%), ST1596 (5.3%), ST11181 (5.3%), ST11750 (2.6/%) and ST11241 (2.6%) among the 38 genotyped isolates. The blaTeM-1B and tetM (55%) was the most prevalent combination of AMR genes, followed by blaTeM-1B (18.4%), tetM (15.8%), and the combination of blaTeM-1B, ermT, and tetL was 2.6% of the isolates. The AMR phenotypes were predicted in ciprofloxacin, penicillin, tetracycline, azithromycin, and cefixime. The combination of mutations 23.7% was gryA (S91F), parC (E91G), ponA (L421) and rpsJ (V57M), followed by 18.4% in gyrA (S91F), ponA (L421P), rpsJ (V57M), and 18.4% in gyrA (D95G, S91F), ponA (L421P), and rpsJ (V57M). The combinations in gyrA (D95G, S91F) and rpsJ (V57M), and gyrA (D95G, S91F), parC (E91F), ponA (L421P) and rpsJ (V57M) were 13.2% each of the isolates. Plasmid TEM-1 (84.2%), tetM (15.8%), and gonococcal genetic island (GGI) was detected in all isolates. Conclusion This study revealed remarkable heterogeneity of N. gonorrhoeae with blaTEM−1, tetM, ponA, gyrA, and parC genes associated with high resistance to penicillin, tetracycline, and ciprofloxacin demanding revision of the standard treatment guidelines and improved antimicrobial stewardship in Zambia. Supplementary Information The online version contains supplementary material available at 10.1186/s12864-024-10155-y.


Background
The sexually transmitted infection (STI) gonorrhea caused by the bacterium Neisseria gonorrhoeae remains a major global public health concern because of its capacity to evolve high levels of resistance to antibiotics available for treatment [1,2].The superbug has developed plasmid-mediated and/or chromosomally mediated antimicrobial resistance (AMR) that has compromised the management of gonorrhea worldwide [3].The AMR mechanisms are usually present in the gonococcal cell and/or a combination of genes with mutations within specific genes to cause resistance to antibiotics [4][5][6].The World Health Organization (WHO) has declared N. gonorrhoeae as a priority pathogen because of its resistance to third-generation cephalosporins (3GS) and fluoroquinolones [7].
In 2020, the World Health Organization (WHO) estimated 82.4 million incident global cases of gonorrhea among adults 15-49 years of age [2].The highest incidence rate of gonorrhea was found in sub-Africa with an increase of 0.2% in women and 1.1% in men every year [19,20].The development of effective vaccines and novel therapeutics would mitigate the emergence and spread of untreatable gonorrhea [21].Gonorrhea can be concomitant with HIV and enhances its transmission [22][23][24][25][26].
The treatment of STIs is according to syndromic management guidelines which have contributed highly to the AMR due to empirical treatment in sub-Sahara Africa [27][28][29][30].The Zambian standard treatment guidelines recommended the use of a single dose of ciprofloxacin in the treatment of gonorrhea (Ciprofloxacin 500 mg PO stat plus doxycycline 100 bd PO X 7/7) [31].The WGS data that provides opportunities to understand the population structure of N. gonorrhoeae for prevention and control of gonorrhea was sparse despite AMR being an emerging phenomenon in Zambia [32].
This study aimed to determine the genetic diversity of N. gonorrhoeae isolated from patients attending STI clinics in urban hospitals in Lusaka, Zambia.

Study Design and Population
A cross-sectional study on 38 Neisseria gonorrhoeae isolated from 122 patients with gonorrhea attending STI clinics in urban hospitals in Lusaka, Zambia.The urethral and endocervical specimens were collected from patients who presented with a discharge from September 2019 to August 2020.The gonococcal isolates were submitted to the University Teaching Hospital (UTH) molecular laboratory for antimicrobial susceptibility testing (AST) and DNA extraction, and to Kilimanjaro Clinical Research Institute (KCRI) at the biotechnology laboratory (KCRI-BL) for molecular testing.The KCRI-BL is GCLP accredited, ISBN 978-1-904610-00-7 operated by Qualogy.

Extraction of genomic DNA
DNA was isolated using the NucliSens easyMaG Nucleic Extraction platform (BioMerieux, Marcy-I'Etoile, France), according to the manufacturer's instructions.A loopful of Neisseria gonorrhoeae from pure cultures grown on chocolate agar (Mast Diagnostics, Merseyside, UK) were transferred into a microcentrifuge tube containing 400µL of 1x TE buffer (10mM Tris-HCl [pH 8.0], 0.1mM EDTA [pH 8.0]) for the bacterial suspension.The bacterial preparation was transferred to the sample strip well of the extractor with the elution of 50µL.The DNA preparation was then preserved at -20 o C before further analysis.

DNA quantification and sequencing
The cgDNA was quantified using a Qubit V4.0 fluorometer (Invitrogen by Thermo Fisher Scientific).The volume of 2µL of DNA was quantified before sequencing.The whole genome sequencing (WGS) was performed using Illumina DNA library preparation protocol, document 1,000,000,025,416 v09 (Illumina Inc., San Diego, CA, USA).Input cgDNA of 100ng was used for library preparation.The amplified DNA library was cleaned using double-sided beads and purified in resuspension buffer (RSB).The paired-ends 150 bp indexed reads were generated using the mid-output protocol on the Illumina Next-seq550 platform (Illumina Inc., San Diego, CA, USA).
The isolates with MLST ST 7363 , ST 1921 , ST 1582 , ST 1588 , ST 1596 , ST 11181 , and ST 11750 were associated with ciprofloxacin, penicillin and tetracycline resistance with a combination of blaTEM-1B and tetM AMR genetic determinants.Phenotypic resistance to tetracycline was detected in isolates carrying tetM and rpsJ genes while 2.6% of the isolates each exhibited AMR phenotype to cefixime and azithromycin (Table 1).The G45A deletion in mtrR promoter was identified in 10.6% (4/38) of the isolates.All the isolates had non-mosaic penA alleles which were associated with susceptibility to ceftriaxone.The identical F504L type II non-mosaic penA allele: 2.002 penicillin MIC 0.5 µg/mL was detected in 66% (25/38), and Type XIX non-mosaic penA allele: 19.001 penicillin MIC 0.5 µg/mL was detected in 34% (13/38) of the isolates.
The multiple sequence alignment CCphylotree was generated using the reference strain WHO O with Gen-Bank accession number NZ_LT592146/GCF_900087625 with MLST sequence type ST 1902 because the majority of the isolates were closest to that reference strain.The multiple sequence alignments included 160 reference genomes for N. gonorrhoeae at assembly levels "Complete Genome" and "Chromosome", retrieved on 17 Oct 2022.The isolates clustered amongst themselves rather than  around the reference strain (Fig. 4).The clades showed inter-clade distances of 5000 SNPs.

Discussion
The AMR of N. gonorrhoeae has been on the increase and is considered a public health challenge in various regions around the world [7].Nowadays, WGS has been used widely in the typing and monitoring of resistant strains of N. gonorrhoeae [53].Data presented in this study showed genetically diverse N. gonorrhoeae having 10 different MLSTs, which includes novel ST 17026 identified in 8% (3/38) of the isolates.The three isolates with novel ST shared a single MLST profile with a novel combination of abcZ:109 adk:39 aroE:67 fumC:771 gdh:148 pdhC:71 pgm:65 [54].The substantial number of the novelty of the isolates corresponds with findings observed in South Africa [55].The ST 7363 , ST 1921 , and ST 1582 representing 55% (21/38) were the most circulating sequence types in Lusaka, Zambia, and highly resistant to ciprofloxacin, penicillin, and tetracycline [33].The combinations of amino substitutions in the gyrA (S91F, D95G) and parC (E91G) were associated with resistance to ciprofloxacin.The study findings are in agreement with studies in Tanzania and South Africa where 70% of the isolates with MLST ST 7363 and ST 1901 were resistant to ciprofloxacin, penicillin, and tetracycline [59,60].Penicillinase-producing N. gonorrhoeae (PPNG) strains 84.2% (32/38) were detected in all the STs, and 2.6% (1/38) of the isolates had reduced susceptibility to cefixime with MIC 0.75 µg/mL (Additional file 1: Table S1).The PPNG isolates are highly prevalent and the global concern is that the bla TEM−1 gene encoding TEM−1 β-lactamase requires few specific single nucleotide polymorphisms (SNPs) to evolve into a gene encoding an extended-spectrum β-lactamase (ESBL) which could degrade all cephalosporins including ceftriaxone [61,62].All isolates harbored the GGI which is a type IV secretion system (T4SS) implicated in AMR to multiple antimicrobials [63].Mutations within the mtrR gene are usually found amongst multidrug-resistant isolates of N. gonorrhoeae and are associated with the outflow of antimicrobials from the bacterial cell [64].Furthermore, 2.6% of the isolates exhibited resistance to azithromycin, and all the isolates were susceptible to ceftriaxone [33].The rise in the number of reports on treatment failures with 3GS demands urgent development of new antimicrobials for the treatment of gonorrhea and increased AMR surveillance remains vital to the prevention and control of gonorrhea worldwide [65-67].

Conclusion
The genomic analysis of the study showed a remarkable genetic diversity of N. gonorrhoeae with bla TEM−1 , tetM, ponA, gyrA, and parC genes associated with high resistance to penicillin, tetracycline and ciprofloxacin demanding for revision of the Zambian standard treatment guidelines.The detection of N. gonorrhoeae resistant to azithromycin demands improved antimicrobial stewardship to prevent an epidemic of untreatable gonorrhea in Zambia.

Limitation of the study
The isolates studied were only from hospitals in Lusaka and might not be representative of other settings in Zambia.

Fig. 3 Fig. 2
Fig. 3 Frequency and percentage of AMR mutations from 38 N. gonorrhoeae isolates

Fig. 1
Fig. 1 Frequency and percentage of sequence types of N. gonorrhoeae

Fig. 4
Fig. 4 MLST phylogenetic tree of 37 Neisseria gonorrhoeae isolates with WHO O reference strain with GenBank accession number NZ_LT592146.1 in Lusaka, Zambia.Symbol key: NOV = Novel sequence types.The scale bar represents the estimated evolutionary divergence of the isolates